Lipopolysaccharide lps from e coli

Mueller-Hinton broth (MHB), Mueller-Hinton agar (MHA) and MRS powder were obtained from AoBoX (China). Bovine serum albumin (BSA), Triton X-100, polymyxin B, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), dimethyl sulfoxide (DMSO), BODIPY-TR-cadaverine (BC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 3,3′-dipropylthiadicarbocyanine (disc_3–5), ethanol (analytical grade, 99%), tertiary butanol (analytical grade, 99%), acetone (analytical grade, 99%), glutaraldehyde (synthetic grade, 50% in H₂O), lipoteichoic acid (LTA) from S. aureus and lipopolysaccharide (LPS) from E. coli were purchased from Sigma-Aldrich (China). Phosphate-buffered saline (PBS) solution, sodium chloride, potassium chloride, ammonium chloride, calcium chloride, zinc chloride, magnesium chloride, and ferric chloride were purchased from Kermel (China). Glucose (analytical grade) was obtained from Zhiyuan (Guangdong, China). DMEM phenol red-free medium and fetal bovine serum were purchased from Gibco (Beijing, China). All these reagents were used according to the required concentration range.

Whole blood from tubes containing lithium heparin were diluted 1:2 with RPMI 1,640 culture medium containing 200 U of penicillin/mL and 200 mg of streptomycin/mL, transferred to 24-well plates, and stimulated with lipoteichoic acid (LTA) from Streptococcus faecalis (final concentration 1 μg/mL, Sigma Aldrich), lipopolysaccharide (LPS) from E. coli 0127:B8 (final concentration, 100 ng/mL, Sigma Aldrich), or phosphate-buffered solution (PBS) as a negative control. Samples were incubated in the dark at 37°C in 5% CO₂ for 24 h. The samples were then centrifuged (400 g for 7 min) and supernatant collected and stored at −80°C for batch analysis. For analysis, samples were thawed, and then tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, IL-8, and monocyte chemoattractant protein (MCP)-1 were quantified with a canine cytokine-specific multiplex bead-based assay (Milliplex MAP, EMD Millipore Corp.) as described elsewhere (16 (link)). The median fluorescence intensity and cytokine concentration in each sample was measured in duplicate with appropriate controls and associated data analysis software (Milliplex Analyst version 5.1, EMD Millipore Corp.). The lower limit of detection for TNF-α, IL-6, IL-10, GM-CSF, IL-2, and MCP-1 was 48.8 ρg/mL. The lower limit of detection for IL-8 was 195.3 ρg/mL.

Human alpha thrombin was obtained from Enzyme Research Laboratories (South Bend, IN). Lipopolysaccharide (LPS) from E. coli, diethylaminoethyl (DEAE)-dextran, and 4-phenylbutyrate (4-PBA) were purchased from Sigma-Aldrich Chemical (St. Louis, MO). Polyclonal antibodies to VCAM-1, ICAM-1, IκBα, RelA/p65, and β-actin were from Santa Cruz Biotechnology (CA). Antibodies to VE-cadherin were purchased from Abcam (Cambridge, MA) and BD Biosciences (San Jose, CA). BiP/GRP78 polyclonal antibody was obtained from Cell Signaling Technology (Beverly, MA). Expression vector encoding Wild type BiP/GRP78 and dominant negative BiP/GRP78 were from Addgene. SubAB, SubA and the non-active derivative SubA_A272B were purified as previously described^{28 (link),29 (link),46 (link)}. All other materials were from VWR Scientific Products Corporation (Gaithersburg, MD) and Fisher Scientific (Pittsburgh, PA).